The long-term goal of this research is to devise a novel and practical tRNA aminoacylation system based on ribozymes, which facilitates the technique of nonnatural amino acid mutagenesis and thus raises it to a more user-accessible technology. An artificial ribozyme selected in our laboratory exhibits a broad spectrum of activities toward phenylalanine (Phe) analogs and suppressor tRNAs bearing different nonsense codons. By immobilizing this ribozyme (called PheFlexizyme) on a resin, the synthesis of suppressor tRNAs charged with Phe analogs is largely facilitated; where a user-specified tRNA and a Phe analog are reacted on this resin and the resulting filtrate (or supernatant) contains the desired aminoacyl-tRNA. This charged tRNA is then used in a cell-free translation system to incorporate a Phe analog at a single position or two Phe analogs at two specific positions. The entire processes, including tRNA charging, in vitro translation, and purification of protein, can be done in one day. The translation efficiency is generally 50-70 mu/g/mL, thus it is feasible to produce a sufficient amount of protein for further biological studies. It should be noted that the PheFlexizyme-resin can be reused more than 10 times, and therefore it is also economical. In this application, we will attempt to develop more sophisticated ribozyme aminoacylation technologies. Specific aims are (1) expanding repertoire of Phe analogs for PheFlexizyme, (2) in vitro evolution of new Flexizymes based on the PheFIlexizyme scaffold, (3) in situ generation of PheFlexizyme in the cell-free transcription-coupled translation apparatus, and (4) applications of PheFlexizyme.